Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 April 2021

Wheat speciation and adaptation: perspectives from reticulate evolution

Authors Xuebo Zhao1,2, Xiangdong Fu1,2, Changbin Yin1,*, Fei Lu1,2,3,*

Affiliations 1State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, China. 2University of Chinese Academy of Sciences, Beijing, China. 3CAS-JIC Centre of Excellence for Plant and Microbial Science (CEPAMS), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

Correspondence [email protected] (F.L.); [email protected] (C.Y.)

Abstract

Reticulate evolution through the interchanging of genetic components across organisms can impact significantly on the fitness and adaptation of species. Bread wheat (Triticum aestivum subsp. aestivum) is one of the most important crops in the world. Allopolyploid speciation, frequent hybridization, extensive introgression, and occasional horizontal gene transfer (HGT) have been shaping a typical paradigm of reticulate evolution in bread wheat and its wild relatives, which is likely to have a substantial influence on phenotypic traits and environmental adaptability of bread wheat. In this review, we outlined the evolutionary history of bread wheat and its wild relatives with a highlight on the interspecific hybridization events, demonstrating the reticulate relationship between species/subspecies in the genera Triticum and Aegilops. Furthermore, we discussed the genetic mechanisms and evolutionary significance underlying the introgression of bread wheat and its wild relatives. An in-depth understanding of the evolutionary process of Triticum species should be beneficial to future genetic study and breeding of bread wheat.

Keywords: wheat; reticulate evolution; introgression; speciation; hybridization; adaptation; breeding

Introduction

The ‘tree of life’ is a classic paradigm representing the evolution of life and illustrating the relationship of species1. While being practical and useful, the tree-like model can be incomplete because fusion, transfer, and exchange of DNA between species can form a net-like evolutionary relationship, shaping the paradigm of reticulate evolution2. Recently, a growing body of research shows that reticulate evolution is common in nature – hybridization, gene flow, and polyploidization were identified in multiple species of humans3–6, animals7–15, and plants16–20. The drastic turnover of genetic components brings forth rapid changes of genetic

© 2021 by the author(s). Distributed under a Creative Commons CC BY license. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 April 2021

diversity, diversification, and speciation, establishing a new fitness landscape of species7,14. For crops, gene flow through reticulate evolution can be a valuable resource to investigate genetic mechanisms and candidate genes of crops adapting to various environments21.

Wheat cultivation began from the Neolithic Age. Over the past few thousand years, wheat was of extreme importance as a staple food that facilitated human civilization development22. In the present-day, wheat has become the second most-produced cereal crop and the most widely grown crop in the world, contributing ~20% of the calories and proteins to the human diet23. However, climate models predict an acceleration of environmental extremes in near future, which are probably the prelude to a subsequent shortage of agricultural supply worldwide24. Moreover, the climate resilience of wheat was found declining in most European countries during the last 10 years, probably due to the reduced diversity in the genetic pool of cultivars25,26. Improving the ability of crops to cope with both biotic and abiotic stresses will likely play an important role in the adaptation of agriculture to climate change in the coming decades27,28. To address the agricultural challenges of this century, there is a pressing demand to understand the adaptive evolution of wheat and its wild relatives, and better utilize the gene pool of wheat.

Reticulate evolution of wheats (Triticum species) has long been recognized. The speciation of wheats is a classic example of reticulate evolution through allopolyploidization, shaping tetraploid and hexaploid species in the genus Triticum29–31. Two economically important wheats are bread wheat (Triticum aestivum subsp. aestivum, AABBDD, 2n = 6x = 42) and durum wheat (T. turgidum subsp. durum, AABB, 2n = 4x = 28), comprising 95% and 5% of the global wheat production, respectively32,33. Research of wheat evolution has focused primarily on two successive rounds of polyploidization events of bread wheat34,35. Homoploid hybridization, introgression, and their biological significance received considerably less attention. Recent studies showed that bread wheat received introgression from their wild relatives, facilitating its adaptation to new environments30,31 and resistance to biotic stress36. The benefits of wild species as readily available resources of adaptive alleles are getting recognized37. Here, we review the process of reticulate evolution of Triticum species, then discuss the biological significance of reticulate evolution in bread wheat and its wild relatives, with a highlight on the impact of introgression upon global adaptation of wheat.

Phylogenetic tree of Triticum species

The Triticum-Aegilops alliance, which contains the direct genetic donors of bread wheat, belongs to the tribe Triticeae. The tribe Triticeae emerging about 25 million years ago (Mya), was split into two subtribes, Hordeineae and Triticineae, about 15 million years ago38,39. Triticineae is an economically important subtribe, giving rise to the domesticated cereals of wheat, rye, and several important forage grass33 (Fig. 1A). Bread wheat and its closely related species (Table 1) include 9 species and 29 subspecies, growing mostly in temperate zones, principally in the northern hemisphere40,41. These subspecies are annuals and have solitary spikelet, with their original habitats in the eastern Mediterranean, and the cradle of agriculture– Fertile Crescent42,43. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 April 2021

Table 1 The nomenclature and main traits of bread wheat and their wild relatives in Triticum and Aegilops Genome Pollination Reference Brittle Free- Names in this review Species Subspecies Breeding state constitution mode* genome rachis threshing (common name) Ae. speltoides Ae. speltoides subsp. speltoides SS C No Wild Yes No Speltoides Tausch Ae. speltoides subsp. ligustica (Savign.) Zhuk. SS C No Wild Yes No Ligustica Ae. tauschii Coss. subsp. strangulata DD S Yes Wild Yes No Strangulata Ae. tauschii Ae. tauschii Coss. subsp. tauschii DD S No Wild Yes No Tauschii T. monococcum L. subsp. aegilopoides (Link) Thell. AA S No Wild Yes No Wild einkorn T. monococcum T. monococcum L. subsp. monococcum AA S No Domesticated No No Domesticated einkorn T. sinskajae T. sinskajae A.Filat. & Kurk. subsp. sinskajae AA S No Domesticated No No Sinskaya T. urartu T. urartu Tumanian ex Gandilyan AA S Yes Wild Yes No Urartu T. turgidum L. subsp. dicoccoides (Korn. ex Asch. & Graebn.) Thell. AABB S Yes Wild No No Wild emmer T. turgidum L. subsp. dicoccon (Schrank) Thell. AABB S No Domesticated No No Domesticated emmer T. karamyschevii NEVSKI var. karamyschevii AABB S No Domesticated No No Georgian wheat T. ispahanicum Heslot AABB S No Domesticated No No Ispahanicum T. turgidum T. turgidum L. subsp. turgidum AABB S No Domesticated No Yes Rivet wheat T. turgidum L. subsp. polonicum (L.) Thell. AABB S No Domesticated No Yes Polish wheat T. turgidum L. subsp. carthlicum (Nevski) A. Love & D. Love AABB S No Domesticated No Yes Persian wheat T. turgidum L. subsp. turanicum (Jakubz.) A. Love & D. Love AABB S No Domesticated No Yes Khorasan wheat T. turgidum L. subsp. durum (Desf.) Husn. AABB S Yes Domesticated No Yes Durum T. timopheevii subsp. armeniacum (Jakubz.) van Slageren AAGG S No Wild Yes No Wild timopheevii T. timopheevii T. timopheevii subsp. timopheevii AAGG S No Domesticated No No Domesticated timopheevii T. aestivum L. subsp. spelta (L.) Thell. AABBDD S Yes Wild/Domesticated No No Spelt T. aestivum L. subsp. macha (Dekapr. & Menabde) Mackey AABBDD S No Wild/Domesticated No No Macha T. aestivum L. subsp. compactum (Host) Mackey AABBDD S No Domesticated No Yes Club wheat T. aestivum L. subsp. sphaerococcum (Percival) MacKey AABBDD S No Domesticated No Yes Indian dwarf wheat T. aestivum T. aestivum subsp. yunanense AABBDD S No Domesticated No No Yunan wheat T. petropavlovskyi Udachin & Migush. AABBDD S No Domesticated No Yes Xinjiang wheat T. aestivum subsp. tibetanum AABBDD S No Wild/Domesticated Yes No Tibetan semi-wild T. vavilovii (Tumanian) Jakubz. var. vavilovii AABBDD S No Wild/Domesticated No Yes Vavilovii T. aestivum L. subsp. aestivum AABBDD S Yes Domesticated No Yes Bread wheat T. zhukovskyi T. zhukovskyi Menabde & Ericz. AAAAGG S No Wild No No Zhukovskyi Source: Wheat Genetics Resource Center (https://www.k-state.edu/wgrc/genetic_resources/triticum_accessions/index.html). * C cross-pollination, S self-pollination

During the speciation of bread wheat, the first hybridization occurred ~0.8 Mya between close relatives (BB) of Ae. speltoides and T. urartu (AA), giving rise to the allotetraploid wild emmer wheat (T. turgidum subsp. dicoccoides, AABB) by polyploidization. At the beginning of agriculture in the Fertile Crescent about 10,000 years ago, wild emmer was domesticated and their cultivated forms appeared44. Domesticated emmer wheat (T. turgidum subsp. dicoccon) was the product of this initial process with the non-brittle rachis trait. Then, domesticated emmer went through the second stage of domestication and free-threshing tetraploids emerged with naked grain phenotype34. As of today’s economically important wheat, durum is a subspecies of free-threshing tetraploids32. Recent studies showed that bread wheat originated through allopolyploidization from the second hybridization event, between free- threshing tetraploids and Ae. tauschii (DD)31. To date, no wild forms of T. aestivum have been identified, indicating that these hexaploid species are innate cultivars45. Bread wheat landraces are clearly divided into Asian and European groups. Almost all bread wheat varieties are derived from the intensive use of European wheat, suggesting that recent dispersal and selection have resulted in a high imbalance in modern germplasm compared to the ancestors of local varieties31,46 (Fig. 1B).

A B PACMAD Zea Secale cereale Sorghum ~10 Mya Poaceae Southern Levant Oryza ~0.8 Mya Northern Levant Wild emmer (AABB) BOP Brachypodium ~10000 BP Domesticated emmer (AABB) Hordeineae Hordeum

Triticeae Psathyrostachys Secale Free-threshing tetraploids (AABB) Triticineae Aegilops Triticum Hexaploid wheat (AABBDD) Asian bread wheat European bread wheat 60 50 40 30 20 10 0 (Mya) Fig.1 Phylogenetic tree showing the evolutionary relationship of Triticum/Aegilops species. A. Phylogeny of Triticeae and its position in the family Poaceae. B. A pruned tree of Triticum species illustrating the process of domestication and speciation of hexaploid wheat. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 April 2021

Reticulated model completes the phylogenetic relationship of wheats

Natural hybridization is defined as the intercross of individuals from two different species or populations of the same species, while introgression or introgressive hybridization describes the incorporation of alleles from one population into another via hybridization followed by continuous backcrossing47. New subspecies formation in the Triticum-Aegilops alliance occurs frequently by polyploidization as well as intra- and inter- introgression via hybridization, leading to a rapid burst of diversification48–50. For example, the speciation of bread wheat is a result of two successive rounds polyploidization (Fig. 2A). There are several reasons that reticulated evolution can happen in Triticum/Aegilops species. First, in the middle and the end of Pleistocene, diploid donors and tetraploid donors formed the allopolyploid species of Triticum by genome duplication via hybridization, possibly due to the global cooling during primary ice ages51,52. Second, being relatively young species, the Triticum-Aegilops alliance showed an exceptional hybridization capacity involving most of its members, most of which have not yet reached complete reproductive isolation at the species level38,50. Third, wheat with different ploidy levels and domestication statuses were grown in the same field. Hybridization and introgression provided a wide range of phenotypes and made possible a selection of desirable genotypes to the farmers. For thousands of years, farmers have been growing mixtures of different genotypes and even different cytotypes53,54. Major hybridization events are as follows.

Hybridization events in the diploid Triticum/Aegilops species

Cultivated wheats are derived from an intricate history of four basic genome types, A, B, G, and D, presented in both diploid and polyploid species. The diploid Triticum and Aegilops species started to diverge from one another during the middle of Pliocene, about 2 to 4 Mya38. The genus Triticum consists of the A-genome diploids T. urartu (2n = 2x = 14, AuAu) and T. monococcum subsp. aegilopoides (2n = 2x = 14, AA). Similar to the diploid Triticum species, the genome of diploid Aegilops species, including Ae. tauschii Coss. (2n = 2x = 14, DD) and Ae. speltoides Tausch (2n = 2x = 14, SS), evolved from the 7 ancestral chromosomes of Triticum-Aegilops alliance55,56. The B genome and G genome in Aegilops might be referred to as S genome in other treatments. Nevertheless, the B, G, and S genomes, though related, are quite distinct40.

Hybridization events raised long-standing controversies in the classification of diploid Triticum/Aegilops species. Marcussen et al.55 and Baidouri et al.57 proposed that the D genome originated from an ancient hybridization event between the A and B lineages about 2.1~2.9 Mya. However, using transcriptome data phylogenomic analysis, Sylvain et al.58 suggested that another wild species Ae. Mutica may be closer to D genome's ancestor than B genome and B genome has continuous introgression into D genome59 (Fig. 2B, label a). In addition, by comparing with the Triticum urartu (AA) and Aegilops tauschii (DD), whether the B genome in bread wheat originated from Ae. speltoides (SS) is also in intense debate. The findings from earlier studies suggested two hypotheses. First, the progenitor of the B genome is a unique and ancient Aegilops species that remains as an unknown ancestor closely related to Ae. speltoides60. Second, the progenitor of the B genome is Ae. speltoides, but the genome of extant Ae. speltoides comprises segments of several species in Aegilops introgressed into Ae. speltoides61. Due to the self-incompatible nature of Ae. speltoides, ongoing genome recombination in its natural population makes the origin of the B genome more bewildering. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 April 2021

Fig.2 The reticulate evolutionary relationship between bread wheat and its progenitors in Triticum/Aegilops species. A. Schematic diagram of the speciation process of bread wheat through two successive rounds polyploidization. B. Model of reticulated evolution involving Triticum/Aegilops species. The diploid, tetraploid, and hexaploid species are separated by gray dotted line. The red arrows show occurrences of allopolyploid hybridization events, which are labeled from 1 to 4. The black arrows show domestication events. The green, blue and orange dotted line show the homoploid hybridization events from diploids, tetraploids and hexaploids, which are labeled from a to k. The areas of blue, green, purple, and orange indicate hulled tetraploids, free-threshing tetraploids, hulled hexaploids, and free-threshing hexaploids, respectively.

Speciation through polyploidization in Triticum

Allopolyploidization is one of the driving forces creating new species62,63. Allotetraploid wheat T. turgidum (genome AABB) originated from hybridization events involving two diploid progenitors classified in the genera Aegilops and Triticum (Fig. 2B, label 1). Genome B, as described above, which was derived from Ae. speltoides or from a closely related species to Ae. speltoides, underwent changes of the ploidy level. Genome A, which has been modified relatively little, was derived from a sister species of T. urartu. Another allotetraploid wheat T. timopheevii (genome AAGG) contains a pair of A genomes from T. urartu and a pair of G genomes55,64 (Fig. 2B, label 2). T. turgidum and T. timopheevii were domesticated, and their cultivated forms appeared during the early stage of agriculture in the Fertile Crescent about Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 April 2021

10,000 years ago. These newly formed tetraploid wheats then went through extensive diversification due to human selection. The evolution of the T. turgidum as crops was initiated when wild emmer was domesticated and formed domesticated emmer wheat (T. turgidum subsp. dicoccon) and its varieties, T. turgidum subsp. karamyschevii and T. turgidum subsp. ispahanicum65,66. As the taxa representing the second stage of domestication, free-threshing tetraploids were derived from domesticated emmer, such as the extinct tetraploid T. turgidum subsp. parvicoccum, appearing in the archaeological record shortly after domesticated emmer67. As the most well-known free-threshing tetraploids, durum wheat is used for macaroni and semolina products32. Most other tetraploid wheat subspecies are free-threshing and probably arose relatively recently, including T. turgidum subsp. turgidum, T. turgidum subsp. turanicum, T. turgidum subsp. polonicum, T. turgidum subsp. carthlicum, all of which are quite similar to durum wheat and differ by only a few traits40. Likewise, the diploid T. monococcum also was domesticated in the same area from wild einkorn (T. monococcum subsp. aegilopoides) to domesticated einkorn (T. monococcum subsp. monococcum)22,43. However, they did not contribute to the speciation of tetraploid species of Triticum through polyploidization.

Speciation through hexaploidization also happens in the genus Triticum. T. aestivum (genome AABBDD) is thought to have arisen through hybridization between T. turgidum and Aegilops tauschii (Fig. 2B, label 3), while T. zhukovskyi (genome AAAAGG) originated through hybridization between T. timopheevii and domesticated einkorn T. monococcum (Fig. 2B, label 4)68–70. Ae. tauschii subsp. strangulata is the accepted ancestor of the D subgenome of bread wheat71, but the tetraploid donor was not clear until a recent study showed that AB subgenomes of bread wheat originated from free-threshing tetraploids31. To date, no wild forms are identified for either T. aestivum or T. zhukovskyi., indicating that these hexaploid species are innate cultivars. Other hexaploid wheats include 3 subspecies based on morphological traits–T. aestivum subsp. compactum, T. aestivum subsp. macha, and T. aestivum subsp. spelta, and 5 subspecies named according to geographical location–T. aestivum subsp. sphaerococcum, T. aestivum subsp. Yunanense, T. aestivum subsp. petropavlovskyi, T. aestivum subsp. tibetanum, and T. aestivum subsp. vavilovii40,41.

Speciation through homoploid hybridization in Triticum

Hybridization creates new species not only through polyploidization but also from homoploid hybridization72,73, in which the chromosome number is not changed in the offspring. In tetraploids, gene flow from wild populations in southern Levant contributed to the population structure of domesticated emmer, but domesticated emmer actually originated from northern Levant65,74 (Fig. 2B, label b). Moreover, T. turgidum subsp. karamyschevii are considered to be a segregant from a hybrid between wild emmer and bread wheat75 (Fig. 2B, label c), and Persian wheat (T. turgidum subsp. carthlicum) may receive the introgression from hexaploid wheats76 (Fig. 2B, label d).

With regards to the origin of hexaploid wheats, a once-popular idea was that spelt wheat was the primitive form of hexaploid wheats because the initial hexaploid wheat was expected to have hulled seed inherited from Ae. tauschii, and therefore would be morphologically similar to spelt77. However, HMW glutenin analysis showed a result of hybridization between T. aestivum and domesticated emmer as the origin of European spelt78–80 (Fig. 2B, label e). Macha wheat (T. aestivum subsp. macha) provides another example of bread wheat diversification through homoploid introgression75 (Fig. 2B, label f), and the genome of Xinjiang wheat (T. subsp. petropavlovskyi) has many introgressed segments from free-threshing tetraploids81 (Fig. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 April 2021

2B, label g). In bread wheat, introgressed segments were found from their diploid and tetraploid progenitors, accounting for a substantial portion of the bread wheat genome74. The AB subgenomes of bread wheat received large amounts of introgression from multiple wild tetraploid populations, such as wild emmer (Fig. 2B, label h), domesticated emmer (Fig. 2B, label i), and free-threshing tetraploids (Fig. 2B, label j). However, relatively few Ae. tauschii introgression (Fig. 2B, label k) was introduced into the D subgenome of bread wheat. To date, there is no evidence showing that T. urartu contributed gene flow to bread wheat31,82. This is possibly due to the low survival rate of unreduced gametes and hybrid offspring, as well as the narrow geographical distribution of ancestral diploid populations41,83. An introgressed segment of 150Mb from wild emmer to bread wheat on the 4A chromosome was identified30. The largest introgression segment was from free-threshing tetraploids, which was on chromosome 2A of the bread wheat genome, covering ~50% of the whole chromosome31.

Methods of introgression detection

Compared with polyploidization with altered chromosome number, introgression segments are more difficult to detect at the homoploid level, because ancestral polymorphisms are shared across many descendant species, which is referred to as incomplete lineage sorting (ILS) and it is challenging to be distinguished from introgression21,84. Furthermore, ancient introgression will dilute over generations85. Whole-genome data maximize the power to detect introgression, even if the introgressed segments are rare in the genome17,86–88. Many useful statistics have been developed to detect introgression.

At the whole-genome level, STRUCTURE89, BEAST90 and phyloNet91 could be used to determine whether hybridization events have occurred. Besides, approximate bayesian computation model such as DIYABC92 estimate introgression rate as a component of broader demographic history. At the locus level, three categories of methods have been developed based on the extent of differentiation, phylogenetic relationships, and demographic history. 93 First, the RNDmin are most used based on Fst and dXY statistics. These differentiation-based approaches can be calculated site by site, dense genome-wide data are not required. However, 94 they do not polarize the introgression events. Second, the f3, f4 statistics , D-statistic or ABBA- BABA test95, HyDe96 and five taxon D statistics97 are based on phylogenetic relationships, which is capable of detecting the location and size of introgression, as well as the direction of gene flow, but they have limited ability to distinguish between introgression and ILS. Third, methods such as F statistics98 and S∗ statistic99, coalescent simulations of specific demographic scenarios can obtain the direction of gene flow, in which the information of phylogeny is not required. This group of methods can distinguish introgression and ILS based on the assumption that introgression haplotypes should be larger than ILS haplotypes because recombination can break down haplotypes over generations100. However, the disadvantage of these methods is that the demographic history of species is relatively complex, leading to inaccurate estimation of ancestral state87. Collectively, introgression detection methods have been successfully applied to several systems5,9,13,21,84.

Role of Introgression in the Adaptation of Wheat

Several types of hybridization events have been detected in wheats suggesting that the reticulate evolution pattern largely complete the hierarchical structure of relationships in Triticum-Aegilops Alliance. Hybridization and introgression had an important role in the Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 April 2021

speciation and diversification of wheats, contributing to the flourishing of the genus Triticum and the unclear boundaries between the subspecies. Notably, domesticated tetraploid and hexaploid wheats exhibit a wider range of genetic plasticity than their progenitors and could adapt themselves to various environments29. By integrating subgenomes previously existing in different environments, polyploid genome brings about a broader range of adaptability of allopolyploid wheat52,101,102. However, the polyploidization events induced severe diversity reduction in the ancestral population of allopolyploid wheat, which was likely hindering its range expansion28,103. Therefore, an in-depth and timely discussion about the role of reticulate evolution in the adaptation of wheats is valuable. As the biological significance of homoploid hybridization and introgression has not been thoroughly studied in wheat, we will use examples from a broad range of species to discuss the role of introgression in the adaptation of wheat in the following section.

Recent studies showed that the tremendous diversity loss of bread wheat, during the bottleneck events of polyploidization and domestication, was mainly compensated by extensive introgression from wild populations30,31,74,82 (Fig. 3A). These introgression events captured a considerable proportion of phenotypic variability of both diploid and tetraploid progenitors, granting an incredible potential of bread wheat adapting to human selection in diverse environments. However, pros and cons of introgression may both exist and impact on the adaptation of wheat. To make the most effective introduction of desirable alien alleles into cultivars, it is necessary to understand the genetic mechanism and evolutionary significance of introgression, especially the benefit and cost of introgressed alleles to the recipient population (Fig. 3B).

Fig.3 The process and genetic mechanisms of introgression between bread wheat and wild populations. A. Simplified demographic model of wheat populations. The size of the branches reflects effective population sizes (Ne). Arrows indicates introgression. B. Genetic mechanisms and effect of introgression in wheats.

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The benefits of introgression

Compared with domesticated crops, wild populations are characterized by high diversity, large effective population size, and higher fitness in the natural environment28,104. Introgression from wild relatives contributes to the divergent adaptation of allopolyploid wheat by substantially increasing its genetic diversity, reducing the deleterious mutation, and expand tolerance to extreme environments14,17,105. When these foreign introgression alleles increase the fitness of the recipient pool is called “adaptive introgression”21 (Fig. 3B label 1). The adaptive introgression can happen in several ways, which are listed as follows.

Increasing the genetic diversity. Introgression increases the genetic diversity by up to two to three orders of magnitude more than mutations106,107. In many crops, such as rice108 and soybean18, introgression from wild populations into cultivated species could increase the diversity of cultivated populations substantially. Many researches have demonstrated that the frequent introgression contributed to the restoration of genetic diversity in bread wheat30,31,74,82. Tetraploid introgression to hexaploid was more frequent and relatively recent, probably due to the higher survival rate of the offspring after homologous hybridization. The tetraploid donors include wild emmer wheat, domesticated emmer wheat, and free-threshing tetraploid wheats, which contributes more than 20% of the bread wheat genome in most cultivars. By contrast, introgression from diploid donor (genome DD) into bread wheat was little, accounting for less than 2% of the bread wheat genome. The imbalance of the introgression from tetraploid and diploid donors is the reason for the asymmetric distribution of nucleotide diversity on the ABD 31,74 subgenomes with πB > πA ≫ πD . More than 60% nucleotide diversity of wild emmer was captured by composite gene flow from tetraploids in bread wheat genome. On the contrary, bread wheat preserved only 14% of the nucleotide diversity of its D subgenome donor, strangulata, which led to the extremely low level of nucleotide diversity in the D subgenome of bread wheat31. Thus, we speculate that the subtle nucleotide diversity differences in A and B subgenomes may also resulted from the different level of gene flow in tetraploid wheats from their ancestral populations T. urartu and Ae. speltoides. In addition to the introgression from the genera Triticum and Aegilops, segments introgressed from barley and rye into bread wheat also contributed to the increase of diversity37,109,110.

Reducing the mutation load. Mutation load refers to the accumulation of deleterious alleles across the genome, which reduces the fitness of species111. The introgression regions from wild populations can result in a reduction in deleterious mutation burden compared with other regions. Since a larger effective population size of wild relatives has the capability to remove deleterious alleles effectively, introgressed alleles from wild relatives can offset the bottleneck effect and increase the selective efficacy112. By quantifying deleterious mutation load using additive or recessive models, the maize population showed a strong negative correlation between mutation load and introgression113. In wheat, introgression of wild emmer wheat showed reduced mutation load in hexaploid landraces and cultivars30.

Resistance to biotic and abiotic stress. The introgressed alleles associated with local adaptation could be the key to evolutionary success to local conditions and expansion around the world82. In potato, introgression is more likely to be highly expressed and stress-induced. The introgressed region contains loci associated with disease resistance, drought tolerance, and heat resistance, suggesting that introgression confers key adaptations for survival and may promote tolerance to new environmental pressures during range expansion114. Introgression regions from wild emmer into bread wheat explained a substantial portion of phenotypic variance for harvest weight, plant height, and flowering time traits, whereas the introgression regions from Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 April 2021

Ae. tauschii are related to disease resistance generally, indicated different roles of the three subgenomes during selection and adaptation30,82. Given that biotic and abiotic stress resistances are complex traits, high-throughput phenotyping should help identify functional introgression to cope with a changing climate115,116.

The costs of introgression

The benefits of introgression certainly increase the genetic diversity and enhance the plasticity of wheat adapting to human selection in different environments. However, every coin has two sides. We also have to be soberly aware that gene flow involving domesticated species is not universally beneficial. Genetic background, demographic history, and environmental factors of both donor and recipient populations can affect the efficacy of introgression breeding. Introgression from wild populations also has negative effects to the population of bread wheat. We will discuss the costs of introgression as follow (Fig. 3B, label 1 and 2).

Outbreeding depression. Genetic differences between the parental species often result in a fitness drop in the hybrids, commonly deemed as “outbreeding depression”117–119. An extreme case of outbreeding depression is hybrid incompatibility, where the hybrid offspring become inviable or sterile120. Probably to ensure the gene dosage balance and mitigate the outbreeding depression, the newly formed allopolyploids usually undergo instantaneous elimination and rearrangements of DNA sequences during the first a few generations103,117. These rearrangements, including sequence deletion, transposons activation or deactivation, as well as pseudogenization, subfunctionalization, and neofunctionalization of genes, are extensive and rapid121–123. While wide hybridization has been used intensively in wheat improvement, these wild hybrids often show reduced grain yield and quality37. Triticale is a synthetic species by crossing wheat and rye with the purpose of combining the characteristics of high yield and high quality of wheat with disease resistance, cold resistance, and high lysine content of rye124. However, low seeding rate and shriveled grain are common traits in octoploid triticale, probably due to the outbreeding depression125. Dosage compensation in plants may provide a genetic mechanism making divergent genomes undergo strong interactions and domination in allopolyploid offspring120,126. In hexaploidy wheat, the Q locus is important to wheat domestication and maintains the phenotype of naked grain. The hyperfunctionalization of 5AQ produced the domestication phenotype in tetraploid wheats (AABB). After strangulata (D genome) joining in, 5Dq in bread wheat became subfunctionalized and showed a reduced gene expression, probably to offset the dosage perturbation caused by the addition of the D subgenome127.

Linkage drag. Linkage drag refers to a phenomenon that undesirable genes are introduced into breeding pool with introgression segments of interests attributed to genetic linkage. Early farmers have made long-term and continuous effort in selecting agronomic traits in wheat in different areas53. The introgression from wild populations did increase genetic diversity of the recipient population, but due to the linkage drag, it often gives rise to weedy phenotypes and thus erode the fruits of thousands of years of domestication and improvement. For example, the famous wheat-rye 1BL.1RS translocation has been extensively used in wheat cultivars because of its remarkable effect of disease-resistance128. However, this introgressed segment also contains genes that reduce the processing and baking quality of wheat129.

How are the existing adaptive alleles in bread wheat are maintained given the introgression and concomitant linkage drag occurs so commonly? The retention of adaptive Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 April 2021

alleles in crop populations is largely governed by selection. Studies have shown that introgression signals from wild relatives into domesticates are reduced around genes responsible for major domestication traits30,113 (Fig. 2B label 2). The locus of wheat domestication gene Q is located in the distal region of 5A chromosome, which is characterized by high gene density and high recombination rate. However, the decrease in genetic diversity was found near the gene Q and no introgression from wild populations was detected in bread wheat, in accordance with the studies mentioned above30,130. Likewise, gene Tg, another locus controlling the free-threshing trait, locates also in a distal chromosome with frequent recombination and alien introgression, but shows reduced introgression only within ~10 Mb flanking region and strong negative selection82,131 (Fig. 3B, label 3).

In addition to selection, recombination rate is an important factor determining the extent of introgression in the recipient population (Fig. 2B, label 3). In natural populations of butterfly, the rate the introgression can be predicted by recombination rate across different species 132. From artificial intraspecies cross experiment in barley, recombination rate variation shaped the patterns of introgression genome-wide133. Moreover, recombination modifiers, especially structural variants, play a critical role in resisting introgression. In sunflower, reduced introgression around a large genomic inversion maintains the combination of adaptive alleles116,134. Similar phenomena have not been reported in wheat but the resistance to introgression due to recombination cold spot may also exist.

Genetic swamping in the wild populations. Genetic swamping mostly refers to a circumstance that gene flow from donor species leads to a gradual replacement of local recipient species by hybrids135. In crops, the gene flow between wild populations and cultivars has been found to be bidirectional and genetic swamping may happen in the wild species. The introduction of nontransgenic imidazolinone‐resistant rice cultivars once helped boost rice yield by 50% in southern Brazil during 2003 to 2014. The herbicide resistant weedy rice started to replace the original weedy rice and evolve around 2006, thus more herbicide is required for weed control– the production cost was tripled at the same period and many farmers in the area went out business136. For maize, a highland wild relative of maize (Zea mays subsp. mexicana) contains about 10% of the genome introgressed from maize17. Gene flow from bread wheat to wild population has not been clear so far, but some of the wild relatives of bread wheat was proposed to be the offspring between bread wheat and wild populations41. Wild populations are extremely valuable as a natural germplasm bank for wheat improvement, the gene flow from cultivars may change the genetic make-up of wild populations or even put some endangered species at the risk of extinction135,137. The genetic effect of introgression from cultivars to the wild needs a thorough assessment.

Input of deleterious mutation. Introgressed alleles from donor to the recipient were sometimes deleterious, especially when the donor population has a very limited effective population size. This effect has been observed in the case of Neanderthal introgression into humans, in which the introgression segments was restricted primarily to nongenic regions because of the high mutation load found within Neanderthal genomes138,139. Hence, to increase efficacy of introgression breeding of wheat, it is crucial to investigate the demographic history of wild donor populations and evaluate their overall biodiversity and fitness.

The fate of introgressed segments

The fate of introgressed segments the recipient population largely depends on recombination rate and selective pressure118. In butterfly, introgression can be predicted by recombination rate Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 April 2021

across different populations132. In human, the extent of introgression is found to be associated with local recombination rate and introgression segments are enriched in highly conserved genomic regions6,87. Both lines of evidence demonstrate the key role of recombination in determining the amount of introgression. Moreover, introgression segments will decrease in size over time because of recombination. The time of introgression is negatively correlated with the size of introgression segment140,141. Once the gene flow was established, selection will act on the introgression segments and decide to a large extent whether these segments will be retained30,87,142. Simply speaking, if introgressed segments were beneficial, they are more likely to have increased allele frequency or even been fixed in the recipient population73,87. If the introgressed segment was deleterious, the segment would be purged by negative selection. Or if the segment is neutral, the allele frequency is likely to be fluctuating87,139. Although the genetic mechanisms determining the fate of introgressed alleles are derived from many different species, the same rules should be equally applying in wheat as well. For example, wide-hybridization between bread wheat and Thinopyrum ponticum was conducted to introduce rust resistance and boost yield in bread wheat. One of the introgression lines, Xiaoyan 6, was quite successful and wild grown in China in 1980s. At present-day, many elite wheat cultivars in China are derivatives of Xiaoyan 6, containing introgression segments of rust resistance143,144.

The application of introgression in wheat breeding

Unlike mutations, gene flow makes it possible to introduce large numbers of new alleles into multiple loci simultaneously, which may be the source of functional alleles and even adaptation to polygenic traits107. The awareness of hybridization and introgression attracted researchers and breeders long ago, and the first successful artificial hybrid wheat was reported in the 19th century. In 1876, the Scottish botanist Stephen Wilson produced a wheat-rye hybrid, a milestone in crossing wheat with related species145. Beginning from the 1920s, improvements in in-vitro tissue culture methods, e.g. embryo rescue, allowed the success of previously unsuccessful hybrid combinations146. Since Sears coined the term "chromosome engineering" in 1972147, approaches from molecular biology have been successively used to exploit the genetic diversity in different gene pools and develop new genetic resources derived from alien introgression148,149.

Some disease-resistance and yield-related genes have been transferred from multiple levels of gene pools into wheat through homoeologous recombination, chromosome translocation and other approaches37. The primary gene pool, which is fully sexually compatible with bread wheat, has the same genomic composition comparing to bread wheat and contains three subgenomes of A, B and D. This gene pool consists of the subspecies in T. aestivum, such as spelt wheat, Indian dwarf wheat, and the varieties of bread wheat landrace37,150. The secondary gene pool comprises the majority of Triticum species and part of the Aegilops speices, which share homologous genomes with bread wheat150,151. For example, Lr47152 and Lr42151 are introgressed into bread wheat from Ae. speltoides and Ae. tauschii. Species in the tertiary gene pool belong to the Triticeae tribe, but do not share homologous genomes with bread wheat37,54,153. Rye and barley and representative speices in the tertiary gene pool. One of most notable examples of introgression breeding in wheat is the wheat-rye translocation 1BL.1RS, in which a genomic region in the short arm of wheat chromosome 1B is replaced by a genomic region in the short arm of rye chromosome 1R to enhance disease resistance of wheat128,154. In addition, Sr52155, Sr59156, Pm56157, Sr44158, and β-glucan CslF 159 are also introgressed to bread wheat from H. villosa, rye, Th. Intermedium and barley. The gene flow from hexaploid and tetraploid was increasing in wheat cultivars released before 1960, Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 April 2021

while alien introgression from Triticeae species had increasing contribution in the gene flow in cultivars released after 1960, which reflects the fact that breeders have spent tremendous effort in introducing align alleles into the wheat gene pool since the Green Revolution 60 years ago153,160. Despite these progresses, introgression breeding is still largely empirical, and more research is needed to achieve precise transfer of favorable alleles and efficient production of improved wheat without compromising other traits46,153. Recently, the high throughput genotyping technology increased the efficiency for the precise identifications of target segments from very large populations30,31,74,160–164. Besides, gene editing can realize the transfer of adaptive genes without cross experiment165,166. We anticipate that the availability of genomic tools and the full understanding of rich genetic resources will facilitate the future introgressions for wheat improvement.

Conclusion

The evolution of bread wheat and its wild relatives is a complex and ongoing process in dynamic environments. The most striking features of this evolution are the frequent hybridization events and extensive introgression that lead to diversification and adaptation 7,73. This reticular pattern provides the genetic basis for blurred boundaries between species and a diverse gene pool of bread wheat. Thus, evaluating the wild introgression existing in the cultivated gene pool is important to understand wild diversity relevant for crop adaptation and inform new breeding strategies21,84.

Review of previous studies has made it clear that many questions remain unsettled regarding the evolution of the wheat. For example, the evolutionary relationship between tetraploids and hexaploids at the subspecies level is unclear. Moreover, due to the short history of bread wheat and large amounts of introgression between bread wheat and its wild progenitors, the conventional taxonomic classification of subspecies in Triticum may overemphasizes their differences. Due to the same reasons, the progenitor-domestication- cultivar model applied in most crops may be oversimplified in wheat. There is probably something having been missed in terms of the origin, duration of domestication, and migration routes because of the ongoing gene flow.

In addition to hybridization history, questions about genetic mechanisms of introgression underneath wheat adaptation need to be addressed. The studies of gene flow in Triticum species at the whole-genome level just got off ground recently31,37,74. By combing with massive data from gene expression167, epigenomics, and regulatory elements profiling168–171, answers to these questions will soon be available. The development of chromosome engineering and genome editing makes it possible to introduce target alleles, multi-gene editing, and directed evolution of protein166,172. We believe an in-depth understanding of genetic rules and biological significance of reticulated evolution of Triticum species will help identify useful agronomic genes, facilitate precision breeding, and address challenges from climate change for wheat.

Acknowledgements Due to limited space we apologize to participants whose excellent work not mentioned here. This work is supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA24020201 and XDA24040102) and the National Natural Science Foundation of China (31970631) to F.L., National Natural Science Foundation of China (31921005) to X.F.

Conflict of interest All the authors have no conflict of interest statement. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 April 2021

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